The aminoacyl-tRNA synthetases comprise a family of twenty enzymes that are essential to every living organism. Each enzyme recognizes a single cognate amino acid and covalently attaches it to the correct tRNA. The "charged" tRNA then transfers the amino acid at the ribosome for specific incorporation into the growing polypeptide chain. The fidelity of protein synthesis is completely dependent on accurate substrate recognition by the tRNA synthetases. Some tRNA synthetases have developed editing mechanisms to correct misactivated amino acids. This proofreading step increases fidelity by as much as two orders of magnitude. However, little is known about the recognition of misactivated amino acids or the hydrolytic cleavage mechanism. This proposal outlines an interdisciplinary research plan combining computational, biochemical, and molecular biology approaches to identify the editing active sites and elucidate the molecular mechanism of the tRNA-dcpendent editing activity using leucyl (Leu-) tRNA synthetase as a novel model. The leucine enzyme editing activities occur by distinct species-specific mechanisms. The E. coli enzyme exhibits a clear "post-transfer" activity that requires transfer of the amino acid to the tRNA for editing, while the yeast enzyme functions by a "pre-transfer" mechanism that hydrolyzes misactivated adenylate intermediates. Emerging hypotheses suggest that the pre- and post-transfer editing active sites may be distinct. Preliminary data are described that identifies the post-transfer editing active site. Subsequent proposed work in Specific Aim I will map and delineate the molecular determinants of this editing active site in E. coli Leu-tRNA synthetase. A series of experiments are also outlined to identify and characterize the pre-transfer editing active site using yeast Leu-tRNA synthetase. In Specific Aim II, a strategic plan is presented to determine which noncognate amino acids threaten the fidelity of Leu-tRNA synthetases such that this enzyme maintains an editing active site. Finally, complementation experiments will test the physiological effects of editing-defective tRNA synthetases on cell viability. A detailed understanding of the tRNA synthetase editing mechanism, particularly those that are species-specific, will benefit ongoing pharmaceutical research that is actively exploring tRNA synthetases as a target for antibiotic development. It will also enable reengineering of tRNA synthetases to activate alternative amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or important tools in medicinal and technological applications.